Name: steps for the autologous ex vivo perfused porcine liver-kidney experiment
Uploaded: 2013-12-18T13:00:00
Description: Watch this Scientific Journal Video about Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment at JoVE.com

We would like to thank Sarah Hosgood from the Department of Transplantation, University Hospitals of Leicester, for technical support with the porcine kidney in this model.

1. Preparation of the Procedure Equipment, Solutions, and Experiment Setup
<ol>
	<li>Equipment: 
	The following equipment is required:
	<ol>
		<li>Dissecting instruments: scissors, scalpels, artery forceps, 6x artery clips.</li>
		<li>4x 12-14F catheters (for portal vein, hepatic artery, bile duct and renal artery), 1x 12F catheter (ureter), suture material (20x Vicryl stitches and Vicryl ties), stitch scissors, 3x infusion sets, ice in large insulated transport container, gloves, a large sterile ...

<ol>
	<li>Gravante, G., Ong, S. L., Metcalfe, M. S., Lloyd, D. M., Dennison, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+porcine+hepatic+arterial+supply,+its+variations+and+their+influence+on+the+extracorporeal+perfusion+of+the+liver.">The porcine hepatic arterial supply, its variations and their influence on the extracorporeal perfusion of the liver.</a> J. Surg. Res. 1, 56-61 (2011).</li><li>Gravante, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+response+of+electrolytic+ablation+in+an+ex+vivo+perfused+liver+model.">Cytokine response of electrolytic ablation in an ex vivo perfused liver model.</a> ANZ J. Surg. , 7-8 (2010).</li><li>Gravante, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+response+to+ischemia/reperfusion+injury+in+an+ex+vivo+perfused+porcine+liver+model.">Cytokine response to ischemia/reperfusion injury in an ex vivo perfused porcine liver model.</a> Transplant Proc. 4, 1107-1112 (2009).</li><li>Gravante, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+histological+changes+following+hepatic+electrolytic+ablation+in+an+ex-vivo+perfused+model.">Patterns of histological changes following hepatic electrolytic ablation in an ex-vivo perfused model.</a> Pathol. Oncol. Res. 4, 1085-1089 (2012).</li><li>Gravante, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+acid-base+balance+during+electrolytic+ablation+in+an+ex+vivo+perfused+liver+model.">Changes in acid-base balance during electrolytic ablation in an ex vivo perfused liver model.</a> Am. J. Surg. 5, 666-670 (2012).</li><li>Chung, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+a+kidney+to+the+normothermic+ex+vivo+perfused+porcine+liver+model+does+not+increase+cytokine+response.">Addition of a kidney to the normothermic ex vivo perfused porcine liver model does not increase cytokine response.</a> J. Artif. Organs. 3, 290-294 (2012).</li><li>Chung, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autologous+normothermic+ex+vivo+perfused+porcine+liver-kidney+model:+improving+the+circuit's+biochemical+and+acid-base+environment.">The autologous normothermic ex vivo perfused porcine liver-kidney model: improving the circuit's biochemical and acid-base environment.</a> Am. J. Surg. 4, 518-526 (2012).</li><li>Yanaga, K., Makowka, L., Lebeau, G., Hwang, R. R., Shimada, M., Kakizoe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+liver+perfusion+and+preservation+system+for+transplantation+research+in+large+animals.">A new liver perfusion and preservation system for transplantation research in large animals.</a> J. Invest. Surg. 3 (1), 65-75 (1990).</li><li>Chung, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autologous+normothermic+ex+vivo+perfused+porcine+liver-kidney+model:+improving+the+circuit's+biochemical+and+acid-base+environment.">The autologous normothermic ex vivo perfused porcine liver-kidney model: improving the circuit's biochemical and acid-base environment.</a> Am. J. Surg. 4, 518-526 (2012).</li><li>Chung, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+a+kidney+to+the+normothermic+ex+vivo+perfused+porcine+liver+model+does+not+increase+cytokine+response.">Addition of a kidney to the normothermic ex vivo perfused porcine liver model does not increase cytokine response.</a> J. Artif. Organs. 3, 290-294 (2012).</li><li>Gravante, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypoxia+due+to+isovolemic+hemodilution+on+an+ex+vivo+normothermic+perfused+liver+model.">Effects of hypoxia due to isovolemic hemodilution on an ex vivo normothermic perfused liver model.</a> J. Surg. Res. 1, 73-80 (2010).</li><li>Butler, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+extracorporeal+porcine+liver+perfusion+for+72+hr.">Successful extracorporeal porcine liver perfusion for 72 hr.</a> Transplantation. 8, 1212-1218 (2002).</li><li>St Peter, S. D., Imber, C. J., Lopez, I., Hughes, D., Friend, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+preservation+of+non-heart-beating+donor+livers+with+normothermic+machine+perfusion.">Extended preservation of non-heart-beating donor livers with normothermic machine perfusion.</a> Br. J. Surg. 5, 609-616 (2002).</li><li>Schon, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation+after+organ+preservation+with+normothermic+extracorporeal+perfusion.">Liver transplantation after organ preservation with normothermic extracorporeal perfusion.</a> Ann. Surg. 1, 114-123 (2001).</li><li>Siedlecki, A., Irish, W., Brennan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+graft+function+in+the+kidney+transplant.">Delayed graft function in the kidney transplant.</a> Am. J. Transplant. 11, 2279-2296 (2011).</li><li>Monbaliu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+graft+nonfunction+and+Kupffer+cell+activation+after+liver+transplantation+from+non-heart-beating+donors+in+pigs.">Primary graft nonfunction and Kupffer cell activation after liver transplantation from non-heart-beating donors in pigs.</a> Liver Transpl. 2, 239-247 (2007).</li><li>Skaro, A. I., Jay, C. L., Ladner, D., Abecassis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+donation+after+cardiac+death+and+donation+after+brain+death--reading+between+the+lines.">Trends in donation after cardiac death and donation after brain death--reading between the lines.</a> Am. J. Transplant. 11, 2390-2391 (2010).</li><li>Brandhorst, D., Iken, M., Bretzel, R. G., Brandhorst, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+storage+in+oxygenated+perfluorodecalin+does+not+restore+post-transplant+function+of+isolated+pig+islets+pre-damaged+by+warm+ischemia.">Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia.</a> Xenotransplantation. 5, 465-470 (2006).</li><li>Lin, Q. Y., Chui, K. K., Rao, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+donor+liver+procurement+with+only+aortic+perfusion.">Rapid donor liver procurement with only aortic perfusion.</a> World. J. Gastroenterol. 6, 884-886 (2001).</li><li>Chung, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+multiorgan+ex+vivo+perfused+model:+results+with+the+porcine+liver-kidney+circuit+over+24+hours.">The development of a multiorgan ex vivo perfused model: results with the porcine liver-kidney circuit over 24 hours.</a> Artif. Organs. 5, 457-466 (2013).</li></ol>

In our group the experience and the results achieved with the ex&#160;vivo perfusion of the liver have been paralleled over the years by concomitant modifications in the retrieval technique adopted. In the last few years our group has achieved considerable experience using an ex vivo porcine model to study the hepatic physiology5,11, new liver ablation techniques2,5, and liver immunology3. Despite the technical challenges derived from an additional organ, corrections of ...

Studying a large animal organ physiology in an artificial environment is challenging. Furthermore, maintaining normal physiological and biochemical parameters for a prolonged period requires a closely monitored perfusion method and model. We have previously described our ex vivo autologous perfused porcine liver model for the study of hepatic physiology and significant progress has been achieved, since we began our liver reperfusion ex vivo model in 2005, in areas such as harvesting technique, understanding hepatic artery anatomical variations1, inflammatory response2,3, histological changes following liver electrolytic ablation (EA)4, and changes in acid base balance during EA5. Unfortunately hyperglycemia and metabolic acidosis require external supplements of insulin and bicarbonate to maintain an accurate physiological environment. Stable blood sugar levels and acid base balance are crucial when studying islet auto-transplantation, especially in the early implantation phase. We have developed a new model incorporating the kidney to clear excess metabolites and improve the environment for the islet transplantation6,7.
Extracorporeal perfusion experiments lasting longer than 24 hr can also be performed, but these require a larger team to perform the experiments8.
Animals used in this study received human care and study protocols were in accordance with the United Kingdom laws. They were euthanized with final exsanguinations during the blood-harvesting procedure according to United Kingdom regulations. Porcine liver and a left kidney were retrieved from animals in the food chain and that would otherwise be discarded. In this way important ethical implications can be overcome and the economic burden is lower than more common in vivo studies. The short duration of liver and kidney perfusion time in this model, lasting from 6-24 hr, has been a major limitation of our studies; this can be improved by minimizing the exposure of the organs to the ischemic injury (cold and warm ischemia).

<table border="0" cellpadding="0" cellspacing="0" width="800">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Epoprostenol sodium (Flolan)</td>
			<td style="width:259px;">Glaxo Smith Kline</td>
			<td style="width:137px;">PL10949/0310</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate 8.4%</td>
			<td>Polyfusor, Fresenius Kabi Ltd</td>
			<td style="width:137px;">PL8828/0043</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefuroxime sodium (Zynacef)</td>
			<td>Flynn Pharma Ltd</td>
			<td style="width:137px;">PL13621/0018</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride 14.7%</td>
			<td>Martindale Pharmaceuticals</td>
			<td style="width:137px;">PL1883/6174R</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin&#160;</td>
			<td style="width:259px;">LEO Laboratories Limited</td>
			<td style="width:137px;">PL0043/0038R</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium taurocholate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:137px;">T4009-25G</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin (Actrapid)</td>
			<td>Novo Nordisk</td>
			<td style="width:137px;">EU/1/02/230/003</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Soltran (preservation solution)</td>
			<td style="width:259px;">Baxter Health Care</td>
			<td style="width:137px;">FKB4708G</td>
			<td style="width:211px;">preservation solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Atraumatic centrifugal pump (Bio-console 560)</td>
			<td style="width:259px;">Medtronic Inc., Minneapolis, Minnesota- United States; custom pack, cardiac surgery division Europe</td>
			<td style="width:137px;"></td>
			<td style="width:211px;"></td>
		</tr>
	</tbody>
</table>

steps for the autologous ex vivo perfused porcine liver-kidney experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Liver Function
The liver function tests remained stable for the first 6 hr. However, there were significant changes present only from the 19th hour of the 24 hr experiment (P&#60;0.001). Factor V and X were progressively diminished during the whole experiment and significant changes were evident from the 5th hour of perfusion for factor V and from the 3rd hour for the factor X onward, (P&#60;0.05; Figure&#160;2)20.
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Watch this Scientific Journal Video about Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment at JoVE.com

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

steps-for-autologous-ex-vivo-perfused-porcine-liver-kidney

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Medicine

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...